32 P thiophosphate preparation

ABSTRACT

32  P-Thiophosphates of the general formula: ##STR1## wherein n =1, 2 or 3 and each M, which may be the same or different, is H or a cation, are prepared by heating H 3   32  PO 4  or a salt thereof with at least an equivalent amount of a thiophosphoryl halide and then treating the reaction product with an aqueous medium to hydrolyse the reaction product and form the  32  P thiophosphate.

The present invention relates to a novel process for the preparation of³² P-thiophosphates.

Our published PCT Patent Application WO90/11289 describes thepreparation of nucleoside thiotriphosphates carrying ³² P in thegamma-thiophosphate group, which comprises reacting a nucleosidediphosphate with a ³² P labelled thiophosphate salt. One problem is thatthe only methods available for the preparation of these ³² Pthiophosphates are time consuming and specific activities and yields areoften low.

We have investigated the reaction of H₃ ³² PO₄ with thiophosphorylhalides and have discovered that ³² P thiophosphates are obtained inhigh yield if the involatile portion of the reaction product ishydrolysed with an aqueous medium, preferably of pH at least 7.5.

Accordingly the present invention provides a novel process for thepreparation of ³² P thiophosphates of the general formula: ##STR2##wherein n =1, 2 or 3 and each M, which may be the same or different, isH or a cation, which comprises heating H₃ ³² PO₄ or a salt thereof withat least an equivalent amount of a thiophosphoryl halide and thentreating the reaction product, after the removal of excess unreactedthiophosphoryl halide, with an aqueous medium, preferably of pH at least7.5, to hydrolyse the reaction product and form the ³² P thiophosphate.

The cation is usually an alkali metal or alkaline earth metal, but ispreferably sodium. The thiophosphoryl halide is preferably chloride orbromide.

In the accompanying Drawing, FIG. 1 illustrates apparatus in which thereaction of the invention may take place.

It is preferred to react the H₃ ³² PO₄ in the form of a salt in whichone or two but not all three of the acidic hydrogens are replaced by acation, preferably an alkali metal cation or a quaternary ammoniumcation. One particularly preferred reactant is LiH₂ ³² PO₄. The use of asalt of this type has the effect of reducing the extent of ³² P exchangeinto reaction by-products e.g. ³² PSCl₃ when the intermediate reactionproduct is hydrolysed and so increases the specific activity of thedesired ³² P thiophosphate product. It is also desirable to wash theinside surfaces of the reactor first with acid e.g. HCl, and then withdeionised water, before the condensation reaction commences, to removesurface bound cations.

The reaction between the thiophosphoryl halide and H₃ ³² PO₄ gives riseto a novel diphosphetane intermediate of the formula: ##STR3## wherein Xis halogen, preferably chloro or bromo, and such diphosphetaneintermediates form a further aspect of the invention.

The aqueous medium is preferably alkaline and has a pH usually in therange 7.5-14 and is preferably buffered at that pH. It is preferred touse a non-phosphate buffer, such as Tricine(N-tris[hydroxymethyl]methylglycine) or a borate buffer, to avoiddilution of the labelled thiophosphate with unlabelled orthophosphate atthe end of the reaction. The hydrolysis of the intermediatediphosphetane reaction product can also be brought about using anaqueous medium of pH less than 7 and treatment of the diphosphetane withan aqueous medium of pH as low as 2.5 for several hours results information of the desired ³² P thiophosphate.

The reaction may be carried out using a large excess of thethiophosphoryl halide in the presence of only a few picomoles of H₃ ³²PO₄, allowing the preparation of a high specific activity product on avery low scale. The reaction is normally carried out in the absence ofsolvent, preferably under anhydrous conditions, using temperaturesbetween 100°-200° C., preferably between 150°-180° C. The reaction iscarried out over preferably at least about 1 hour, but usually over 12to 60 hours.

Where the reaction product is not a salt in which all 3M groups arecations, it is preferred to convert the reaction product to such a salt,each cation preferably being an alkali metal e.g., sodium, as such saltsare preferred for reaction with the nucleoside diphosphate in theprocess of the above-mentioned W090/11289.

The following Example illustrates the present invention.

Preparation of High-specific Activity ³² P-labelled Sodium Thiophosphate

A solution of "carrier-free" H₃ ³² PO₄ (2μCi, ca 0.2 p moles, PBK 8855,Amersham International) in water (10 μl) was introduced to the bottom ofthe sidearm A (in the apparatus of FIG. 1) with a plastic tippedpipette. The contents of sidearm A were then frozen with liquid nitrogenand the vessel evacuated to 0.5 mm mercury while connected to a P₂ O₅drying trap. After half an hour, the drying of the H₃ ³² PO₄ wascompleted by heating it, still under vacuum, at 150° C. for one hour asilicone oil bath. The vessel was then cooled, dry nitrogen admitted andthiophosphoryl chloride (10 μl) introduced to the bottom of the sidearmA while flushing gently with dry nitrogen. This was then sealed byclosing the stopcock and the whole of both sidearms was then immersed insilicone oil at 180° C. for 60 hours. Both limbs were then refrozen inliquid nitrogen, the stopcock opened and the apparatus evacuated to 0.5mm mercury. The apparatus was then resealed and sidearm A allowed towarm to room temperature while sidearm B was immersed in liquidnitrogen. This resulted in distillation of all the free thiophosphorylchloride containing 1-2% of exchanged ³² P into B. The residue,consisting of the involatile cyclic derivative1-oxa-3-thia-2,2,2-trichloro-4,4,4-trihydroxy-2,4-diphosphetane andrelated condensed products, was dissolved in Tricine buffer (100 μl, 100mM) at pH 8.0 containing dithio-threitol (10 mM) which led to therelease of thiophosphate ions. A sample (20 μl) was run on apolyethyleneimine/cellulose TLC plate (E. Merck Art. 5579) in 1M sodiumformate at pH 3.4 Radioactivity was associated with the slow-runningblue spot (ammonium molybdate spray) derived from a sample ofnon-radioactive sodium thiophosphate (Rf ca 0.3) (Alfa Inorganics). Ourbatch of commercial material also contained a significant proportion(ca. 30%) of sodium orthophosphate, evidenced by a faster running yellowspot (Rf ca. 0.7).

When the solution of the diphosphetane in aqueous buffer (10 μl) ishydrolysed with concentrated hydrochloric acid (10 μl) at 40° C. for 20minutes and the product is run in the same TLC system, all theradioactivity runs at Rf ca. 0.7, indicating that, under acidconditions, all the thiophosphate had been hydrolysed to orthophosphate.##STR4##

The ³² P-labelled sodium thiophosphate prepared in this way isaccompanied by one equivalent of non-radioactive sodium orthophosphate.This can either be removed by TLC procedures as outlined above, or theproduct used directly to prepare ³² P-labelled adenosinethiotriphosphate, when the adenosine triphosphate formed can be removedfor example by column chromatography on diethylaminoethylsepharose.

We claim:
 1. A process for the preparation of a ³² P thiophosphate ofthe general formula: ##STR5## wherein n=1, 2 or 3 and each M, which maybe the same or different, is H or a cation, which comprises heating H₃³² PO₄ or a salt thereof with at least an equivalent amount of athiophosphoryl halide and then treating the reaction product with anaqueous medium to hydrolyse the reaction product and form the ³² Pthiophosphate.
 2. A process according to claim 1 wherein the pH of theaqueous medium is at least 7.5.
 3. A process according to claim 1wherein the aqueous medium is buffered at the desired pH.
 4. A processaccording to claim 1 wherein the thiophosphoryl halide is reacted withan alkali metal dihydrogen ³² P-orthophosphate.
 5. A process accordingto claim 4 wherein the orthophosphate is LiH₂ ³² PO₄.
 6. A processaccording to claim 1 wherein the reaction product is one in which each Mis an alkali metal or an alkaline earth metal cation or wherein thereaction product is converted to one in which each M is an alkali metalor alkaline earth metal cation.
 7. A process according to claim 6wherein the cation is sodium.
 8. A process according to claim 1 whereinthe heating is carried out at from 150° to 180° C. for 1 to 60 hours. 9.A diphosphetane of the formula: ##STR6## wherein X is halogen.
 10. Adiphosphetane according to claim 9 wherein the halogen is chloro orbromo.
 11. A process for the preparation of a diphosphetane as definedin claim 9 which comprises heating H₃ ³² PO₄ or a salt thereof with atleast an equivalent amount of a thiophosphoryl halide.
 12. A processaccording to claim 11 wherein the heating is carried out at 150° to 180°C. for 1 to 60 hours.